O6 meg

AOM, guanase, O⁶-meG, N⁷-methylguanine (N⁷-meG), 8-azaa-denine were purchased from Sigma Chemical Co. (St Louis, MO, USA). Other chemicals of reagent grade or higher were purchased from Wako Chemicals (Osaka, Japan).

MGMT methyltransferase activity was determined by MGMT assay kit (MD0100, Sigma-Aldrich). Briefly, scrambled control (SC) and KDM6B KO2 HeLa cells were pretreated with vehicle or BG (200 μM) for 30 min and lysed in the modified lysis buffer (50 mM Tris, pH 7.5, 1mM EDTA, 1 mM DTT, 5% Glycerol, 50 mM NaCl). The resulting lysates were incubated with a 23-bp customized biotin-labeled DNA substrate containing an O⁶ MeG next to the PstI cleavage site (Sigma) for 2 h at 37°C. Then the reaction was stopped at 65°C for 5 min. The resulting DNA purified via phenol/chloroform/isoamyl alcohol (25:24:1) with 10 μg yeast tRNA serving as a carrier was incubated with PstI at 37°C for 1 h (10 μl reaction system). The reaction was stopped by 5 μl FBXE buffer (90% Formamide, 0.1% (w/v) Bromophenol blue, 0.1% xylene cyanole, 20 mM EDTA), heated at 95°C for 5 min and quickly chilled on ice, followed by separation on 20% 7 M urea SDS-PAGE gel and immunoblot assay with anti-Biotin antibody (Thermo Fisher Scientific, 692033).